Genetic products differentially expressed in tumors and use thereof

ABSTRACT

The invention relates to the identification of genetic products that are expressed in association with a tumor and the nucleic acid coding therefor. The invention relates to the therapy and diagnosis of diseases in which said genetic products that are expressed in association with a tumor are expressed in aberrant manner. The invention also relates to proteins, polypeptides, and peptides which are expressed in association with a tumor and the nucleic acids coding therefor.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/197,960, filed on Aug. 25, 2008, now U.S. Pat. No.8,716,455, which issued May 6, 2014, which is a continuation applicationof U.S. patent application Ser. No. 10/506,443, filed Sep. 2, 2004, nowU.S. Pat. No. 7,429,461, which issued Sep. 30, 2008 and which is aNational Stage Entry of PCT/EP03/02556, which was filed on Mar. 12, 2003and claims priority to German Patent Application Number 102-11-088.3,which was filed on Mar. 13, 2002, all of which are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

This application includes a sequence listing, which is submitted uponfiling in electronic form only (computer readable “.txt” file), andwhich is incorporated herein by reference in lieu of the paper copy ofthe sequence listing. The content of the electronic sequence listing andthe paper copy of the sequence listing are the same.

BACKGROUND OF THE INVENTION

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath. More recent therapeutic concepts aim at incorporating thepatient's immune system into the overall therapeutic concept by usingrecombinant tumor vaccines and other specific measures such as antibodytherapy. A prerequisite for the success of such a strategy is therecognition of tumor-specific or tumor-associated antigens or epitopesby the patient's immune system whose effector functions are to beinterventionally enhanced. Tumor cells biologically differ substantiallyfrom their nonmalignant cells of origin. These differences are due togenetic alterations acquired during tumor development and result, interalia, also in the formation of qualitatively or quantitatively alteredmolecular structures in the cancer cells. Tumor-associated structures ofthis kind which are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens. Thespecific recognition of tumor-associated antigens involves cellular andhumoral mechanisms which are two functionally interconnected units: CD4⁺and CD8⁺ T lymphocytes recognize the processed antigens presented on themolecules of the MHC (major histocompatibility complex) classes II andI, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens. The potentialclinical-therapeutical importance of tumor-associated antigens resultsfrom the fact that the recognition of antigens on neoplastic cells bythe immune system leads to the initiation of cytotoxic effectormechanisms and, in the presence of T helper cells, can cause eliminationof the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, acentral aim of tumor immunology is to molecule/ly define thesestructures. The molecular nature of these antigens has been enigmaticfor a long time. Only after development of appropriate cloningtechniques has it been possible to screen cDNA expression libraries oftumors systematically for tumor-associated antigens by analyzing thetarget structures of cytotoxic T lymphocytes (CTL) (van der Bruggen atal., Science 254:1643-7, 1991) or by using circulating autoantibodies(Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To thisend, cDNA expression libraries were prepared from fresh tumor tissue andrecombinantly expressed as proteins in suitable systems Immunoeffectorsisolated from patients, namely CTL clones with tumor-specific lysispatterns, or circulating autoantibodies were utilized for cloning therespective antigens.

In recent years a multiplicity of antigens have been defined in variousneoplasias by these approaches. The class of cancer/testis antigens(CTA) is of great interest here. CTA and genes encoding them(cancer/testis genes or CTG) are defined by their characteristicexpression pattern [Tureci et al, Mol Med Today. 3:342-9, 1997]. Theyare not found in normal tissues, except testis and germ cells, but areexpressed in a number of human malignomas, not tumor type-specificallybut with different frequency in tumor entities of very different origins(Chen & Old, Cancer J. Sci. Am. 5:16-7, 1999). Serum reactivitiesagainst CTA are also not found in healthy controls but only in tumorpatients. This class of antigens, in particular owing to its tissuedistribution, is particularly valuable for immunotherapeutic projectsand is tested in current clinical patient studies (Marchand at al., Int.J. Cancer 80:219-30, 1999; Knuth et al., Cancer Chemother. Pharmacol.46:p 46-51, 2000).

However, the probes utilized for antigen identification in the classicalmethods illustrated above are immunoeffectors (circulatingautoantibodies or CTL clones) from patients usually having alreadyadvanced cancer. A number of data indicate that tumors can lead, forexample, to tolerlzation and anergization of T cells and that, duringthe course of the disease, especially those specificities which couldcause effective immune recognition are lost from the immunoeffectorrepertoire. Current patient studies have not yet produced any solidevidence of a real action of the previously found and utilizedtumor-associated antigens. Accordingly, it cannot be ruled out thatproteins evoking spontaneous immune responses are the wrong targetstructures.

BRIEF SUMMARY OF THE INVENTION

It was the object of the present invention to provide target structuresfor a diagnosis and therapy of cancers.

According to the invention, this object is achieved by the subjectmatter of the claims.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1: Diagrammatic representation of the cloning of eCT. The strategycomprises identifying candidate genes (GOI=“Genes of interest”) indatabases and testing said genes by means of RT-PCR.

FIG. 2: Splicing of LDH C. Alternative splicing events result in theabsence of exon 3 (SEQ ID NO:2), of the two exons 3 and 4 (SEQ ID NO:3),of the exons 3, 6 and 7 (SEQ ID NO:4) and of exon 7 (SEQ ID NO:5).ORF=open reading frame, as =amino acid.

FIG. 3: Alignment of possible LDH-C proteins. SEQ ID NO:8 and SEQ IDNO:10 are truncated portions of the prototype protein (SEQ ID NO:6). Theprotein sequences of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEC IDNO:12 and SEQ ID NO:13 are additionally altered and contain onlytumor-specific epitopes (printed in bold type). The catalytic centre isframed.

FIG. 4: Quantification of LDH C in various tissues by means of real timePCR. No transcripts were detected in normal tissues other than testis,but significant levels of expression were detected in tumors.

FIG. 5: Exon composition of TPTE variants. According to the invention,splice variants were identified (SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57) which are expressed intesticular tissues and in tumors and which have frame shifts and thusaltered sequence regions.

FIG. 6: Alignment of the possible TPTE proteins. Alternative splicingevents result in alterations of the encoded proteins, with the readingframe being retained in principle. The putative transmembrane domainsare printed in bold type, the catalytic domain is framed.

FIG. 7: Alignment of TSBP variants at the nucleotide level. Thedifferences in the nucleotide sequences of the TSBP variants foundaccording to the invention (SEQ) ID NO:31, SEQ ID NO:32, SEQ ID NO:33)to the known sequence (NM_006781, SEQ ID NO: 29) are printed in boldtype.

FIG. 8: Alignment of TSBP variants at the protein level. In the proteinsencoded by the TSBP variants found according to the invention (SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36), frame shifts cause substantialdifferences to the previously described protein (SEQ ID NO:30,NM_006781) and are indicated by bold type.

FIG. 9: RT-PCR for MS4Al2. Expression was detected in the tissues testedonly in testis, colon and colorectal carcinomas (colon ca's). In one ofthe 6 liver tissue samples shown, a positive detection was carried outfor MS4Al2, since this sample has been infiltrated by a colon carcinomametastasis. Later studies also demonstrated distinct expression in coloncarcinoma metastases.

FIG. 10: RT-PCR for BRC01. BRCO1 is distinctly overexpressed in breasttumors in comparison with expression in normal mammary gland tissue.

FIG. 11: RT-PCR for MORC, TPX1, LDHC, SGY-1. A study of various normaltissues reveals expression only in testis (1 skin, 2 small intestine, 3colon, 4 liver, 5 lung, 6 stomach, 7 breast, 8 kidney, 9 ovary, 10prostate, 11 thyroid, 12 leukocytes, 13 thymus, 14 negative control, 15testis). The examination of tumors (1-17 lung tumors, 18-29 melanomas,30 negative control, 31 testis) reveals ectopic expression in saidtumors with different frequencies for the individual eCT.

FIG. 12: Mitochondrial localization of LDHC in the MCF-7 breast cancercell line. MCF-7 cells were transiently transfected with an LDHCexpression plasmid. The antigen was detected with LDHC-specificantibodies and showed distinct colocalization with the mitochondrialrespiratory chain enzyme cytochrome C-oxidase.

FIG. 13: Predicted topology of TPTE and subcellular localization on thecell surface of MCF-7 cells. The diagram on the left-hand side depictsthe 4 putative TPTE transmembrane domains (arrows). MCF-7 cells weretransiently transfected with a TPTE expression plasmid. The antigen wasdetected using TPTE-specific antibodies and showed distinctcolocalization with MHC I molecules located on the cell surface.

FIG. 14: MS4Al2 localization on the cell membrane. Tumor cells weretransiently transfected with a GFP-tagged MS4Al2 construct and showedcomplete colocalization with plasma membrane markers in confocalimmunofluorescence microscopy.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, a strategy for identifying and providingantigens expressed in association with a tumor and the nucleic acidscoding therefor was pursued. This strategy is based on the fact thatactually testis- and thus germ cell-specific genes which are usuallysilent in adult tissues are reactivated in tumor cells in an ectopic andforbidden manner. First, data mining produces a list as complete aspossible of all known testis-specific genes which are then evaluated fortheir aberrant activation in tumors by expression analyses by means ofspecific RT-PCR. Data mining is a known method of identifyingtumor-associated genes. In the conventional strategies, however,transcriptoms of normal tissue libraries are usually subtractedelectronically from tumor tissue libraries, with the assumption that theremaining genes are tumor-specific (Schmitt et al., Nucleic Acids Res.27:4251-60, 1999; Vasmatzis et al., Proc. Natl. Acad. Sci. USA.95:300-4, 1998. Scheurle et al., Cancer Res. 60:4037-43, 2000).

The concept of the invention, which has proved much more successful,however, is based on utilizing data mining for electronically extractingall testis-specific genes and then evaluating said genes for ectopicexpression in tumors.

The invention thus relates in one aspect to a strategy for identifyinggenes differentially expressed in tumors. Said strategy combines datamining of public sequence libraries (“in silico”) with subsequentevaluating laboratory-experimental (“wet bench”) studies.

According to the invention, a combined strategy based on two differentbioinformatic scripts enabled new members of the cancer/testis (CT) geneclass to be identified. These have previously been classified as beingpurely testis-, germ cell- or sperm-specific. The finding that thesegenes are aberrantly activated in tumor cells allows them to be assigneda substantially new quality with functional implications. According tothe invention, these tumor-associated genes and the genetic productsencoded thereby were identified and provided independently of animmunogenic action.

The tumor-associated antigens identified according to the invention havean amino acid sequence encoded by a nucleic; acid which is selected fromthe group consisting of (a) a nucleic acid which comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 1-5,19-21, 29, 31-33, 37, 39, 40, 54-57, 62, 63, 70, 74, 85-88, a part orderivative thereof, b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c). In a preferred embodiment, a tumor-associated antigen identifiedaccording to the invention has an amino acid sequence encoded by anucleic acid which is selected from the group consisting of SEQ ID NOs:1-5, 19-21, 29, 31-33, 37, 39, 40, 54-57, 62, 63, 70, 74, 85-88. In afurther preferred embodiment, a tumor-associated antigen identifiedaccording to the invention comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 6-13, 14-18, 22-24, 30, 34-36,38, 41, 58-61, 64, 65, 71, 75, 80-84, 89-100, a part or derivativethereof.

The present invention generally relates to the use of tumor-associatedantigens identified according to the invention or of parts thereof, ofnucleic acids coding therefor or of nucleic acids directed against saidcoding nucleic acids or of antibodies directed against thetumor-associated antigens identified according to the invention or partsthereof for therapy and diagnosis. This utilization may relate toindividual but also to combinations of two or more of these antigens,functional fragments, nucleic acids, antibodies, etc., in one embodimentalso in combination with other tumor associated genes and antigens fordiagnosis, therapy and progress control.

Preferred diseases for a therapy and/or diagnosis are those in which oneor more of the tumor-associated antigens identified according to theinvention are selectively expressed or abnormally expressed.

The invention also relates to nucleic acids and genetic products whichare expressed in association with a tumor cell and which are produced byaltered splicing (splice variants) of known genes or by alteredtranslation with utilization of alternative open reading frames. Saidnucleic acids comprise the sequences according to (SEQ ID NO: 2-5, 20,21, 31-33, 54-57, 85-88) of the sequence listing. Furthermore, thegenetic products comprise sequences according to (SEQ ID NO: 7-13, 23,24, 34-36, 58-61, 89-100) of the sequence listing. The splice variantsof the invention can be used according to the invention as targets fordiagnosis and therapy of neoplastic diseases.

Very different mechanisms may cause splice variants to be produced, forexample

-   -   utilization of variable transcription initiation sites    -   utilization of additional exons    -   complete or incomplete splicing out of single or two or more        exons,    -   splice regulator sequences altered via mutation (deletion or        generation of new donor/acceptor sequences),    -   incomplete elimination of intron sequences.

Altered splicing of a gene results in an altered transcript sequence(splice variant). Translation of a splice variant in the region of itsaltered sequence results in an altered protein which may be distinctlydifferent in the structure and function from the original protein.Tumor-associated splice variants may produce tumor-associatedtranscripts and tumor-associated proteins/antigens. These may beutilized as molecular markers both for detecting tumor cells and fortherapeutic targeting of tumors. Detection of tumor cells, for examplein blood, serum, bone marrow, sputum, bronchial lavage, bodilysecretions and tissue biopsies, may be carried out according to theinvention, for example, after extraction of nucleic acids by PCRamplification with splice variant-specific oligonucleotides. Accordingto the invention, all sequence-dependent detection systems are suitablefor detection. These are, apart from PCR, for example genechip/microarray systems, Northern blot, RNAse protection assays (RDA)and others. All detection systems have in common that detection is basedon a specific hybridization with at least one splice variant-specificnucleic acid sequence. However, tumor cells may also be detectedaccording to the invention by antibodies which recognize a specificepitope encoded by the splice variant. Said antibodies may be preparedby using for immunization peptides which are specific for said splicevariant. Suitable for immunization are particularly the amino acidswhose epitopes are distinctly different from the variant(s) of thegenetic product, which is (are) preferably produced in healthy cells.Detection of the tumor cells with antibodies may be carried out here ona sample isolated from the patient or as imaging with intravenouslyadministered antibodies. In addition to diagnostic usability, splicevariants having new or altered epitopes are attractive targets forimmunotherapy. The epitopes of the invention may be utilized fortargeting therapeutically active monoclonal antibodies or T lymphocytes.In passive immunotherapy, antibodies or T lymphocytes which recognizesplice variant-specific epitopes are adoptively transferred here. As inthe case of other antigens, antibodies may be generated also by usingstandard technologies (immunization of animals, panning strategies forisolation of recombinant antibodies) with utilization of polypeptideswhich include these epitopes. Alternatively, it is possible to utilizefor immunization nucleic acids coding for oligo- or polypeptides whichcontain said epitopes. Various techniques for in vitro or in vivogeneration of epitope-specific T lymphocytes are known and have beendescribed in detail, for example (Kessler J H, et al. 2001, Sahin etal., 1997) and are likewise based on utilizing oligo- or polypeptideswhich contain the splice variant-specific epitopes or nucleic acidscoding for said oligo- or polypeptides. Oligo- or polypeptides whichcontain the splice variant-specific epitopes or nucleic acids coding forsaid polypeptides may also be used for utilization as pharmaceuticallyactive substances in active immunotherapy (vaccination, vaccinetherapy).

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which recognizes the tumor-associated antigenidentified according to the invention and which is preferably selectivefor cells which have expression or abnormal expression of atumor-associated antigen identified according to the invention. Inparticular embodiments, said agent may cause induction of cell death,reduction in cell growth, damage to the cell membrane or secretion ofcytokines and preferably have a tumor-inhibiting activity. In oneembodiment, the agent is an antisense nucleic acid which hybridizesselectively with the nucleic acid coding for the tumor-associatedantigen. In a further embodiment, the agent is an antibody which bindsselectively to the tumor-associated antigen, in particular acomplement-activated antibody which binds selectively to thetumor-associated antigen. In a further embodiment, the agent comprisestwo or more agents which each selectively recognize differenttumor-associated antigens, at least one of which is a tumor-associatedantigen identified according to the invention. Recognition needs not beaccompanied directly with inhibition of activity or expression of theantigen. In this aspect of the invention, the antigen selectivelylimited to tumors preferably serves as a label for recruiting effectormechanisms to this specific location. In a preferred embodiment, theagent is a cytotoxic T lymphocyte which recognizes the antigen on an HLAmolecule and lyses the cell labeled in this way. In a furtherembodiment, the agent is an antibody which binds selectively to thetumor-associated antigen and thus recruits natural or artificialeffector mechanisms to said cell. In a further embodiment, the agent isa T helper lymphocyte which enhances effector functions of other cellsspecifically recognizing said antigen.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which inhibits expression or activity of atumor-associated antigen identified according to the invention, In apreferred embodiment, the agent is an antisense nucleic acid whichhybridizes selectively with the nucleic acid coding for thetumor-associated antigen. In a. further embodiment, the agent is anantibody which binds selectively to the tumor-associated antigen, In afurther embodiment, the agent comprises two or more agents which eachselectively inhibit expression or activity of different tumor-associatedantigens, at least one of which is a tumor-associated antigen identifiedaccording to the invention.

The invention furthermore relates to a pharmaceutical composition whichcomprises an agent which, when administered, selectively increases theamount of complexes between an HLA molecule and a peptide epitope fromthe tumor-associated antigen identified according to the invention, Inone embodiment, the agent comprises one or more components selected fromthe group consisting of (i) the tumor-associated antigen or a partthereof, (ii) a nucleic acid which codes for said tumor-associatedantigen or a part thereof, (iii) a host cell which expresses saidtumor-associated antigen or a part thereof, and (iv) isolated complexesbetween peptide epitopes from said tumor-associated antigen and an MHCmolecule. In one embodiment, the agent comprises two or more agentswhich each selectively increase the amount of complexes between MHCmolecules and peptide epitopes of different tumor-associated antigens,at least one of which is a tumor-associated antigen identified accordingto the invention.

The invention furthermore relates to a pharmaceutical composition whichcomprises one or more components selected from the group consisting of(i) a tumor-associated antigen identified according to the invention ora part thereof, (ii) a nucleic acid which codes for a tumor-associatedantigen identified according to the invention or for a part thereof,(iii) an antibody which binds to a tumor-associated antigen identifiedaccording to the invention or to a part thereof, (iv) an antisensenucleic acid which hybridizes specifically with a nucleic acid codingfor a tumor-associated antigen identified according to the invention,(v) a host cell which expresses a tumor-associated antigen identifiedaccording to the invention or a part thereof, and (vi) isolatedcomplexes between a tumor-associated antigen identified according to theinvention or a part thereof and an HLA molecule.

A nucleic acid coding for a tumor-associated antigen identifiedaccording to the invention or for a part thereof may be present in thepharmaceutical composition in an expression vector and functionallylinked to a promoter.

A host cell present in a pharmaceutical composition of the invention maysecrete the tumor-associated antigen or the part thereof, express it onthe surface or may additionally express an HLA molecule which binds tosaid tumor-associated antigen or said part thereof. In one embodiment,the host cell expresses the HLA molecule endogenously. In a furtherembodiment, the host cell expresses the HLA molecule and/or thetumor-associated antigen or the part thereof in a recombinant manner.The host cell is preferably nonproliferative. In a preferred embodiment,the host cell is an antigen-presenting cell, in particular a dendriticcell, monocyte or a macrophage.

An antibody present in a pharmaceutical composition of the invention maybe a monoclonal antibody. In further embodiments, the antibody is achimeric or humanized antibody, a fragment of a natural antibody or asynthetic antibody, all of which may be produced by combinatorytechniques. The antibody may be coupled to a therapeutically ordiagnostically useful agent.

An antisense nucleic acid present in a pharmaceutical composition of theinvention may comprise a sequence of 6-50, in particular 10-30, 15-30and 20-30, contiguous nucleotides of the nucleic acid coding for thetumor-associated antigen identified according to the invention.

In further embodiments, a tumor-associated antigen, provided by apharmaceutical composition of the invention either directly or viaexpression of a nucleic acid, or a part thereof binds to MHC moleculeson the surface of cells, said binding preferably causing a cytolyticresponse and/or inducing cytokine release.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier and/or an adjuvant. The adjuvant maybe selected from saponin, GM-CSF, CpG nucleotides, RNA, a cytokine or achemokine. A pharmaceutical composition of the invention is preferablyused for the treatment of a disease characterized by selectiveexpression or abnormal expression of a tumor-associated antigen. In apreferred embodiment, the disease is cancer.

The invention furthermore relates to methods of treating or diagnosing adisease characterized by expression or abnormal expression of one ofmore tumor-associated antigens. In one embodiment, the treatmentcomprises administering a pharmaceutical composition of the invention.

In one aspect, the invention relates to a method of diagnosing a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention. The method comprisesdetection of (i) a nucleic acid which codes for the tumor-associatedantigen or of a part thereof and/or (ii) detection of thetumor-associated antigen or of a part thereof, and/or (iii) detection ofan antibody to the tumor-associated antigen or to a part thereof and/or(iv) detection of cytotoxic or T helper lymphocytes which are specificfor the tumor-associated antigen or for a part thereof in a biologicalsample isolated from a patient. In particular embodiments, detectioncomprises (i) contacting the biological sample with an agent which bindsspecifically to the nucleic acid coding for the tumor-associated antigenor to the part thereof, to said tumor-associated antigen or said partthereof, to the antibody or to cytotoxic or T helper lymphocytesspecific for the tumor-associated antigen or parts thereof, and (ii)detecting the formation of a complex between the agent and the nucleicacid or the part thereof, the tumor-associated antigen or the partthereof, the antibody or the cytotoxic or T helper lymphocytes. In oneembodiment, the disease is characterized by expression or abnormalexpression of two or more different tumor-associated antigens anddetection comprises detection of two or more nucleic acids coding forsaid two or more different tumor-associated antigens or of partsthereof, detection of two or more different tumor-associated antigens orof parts thereof, detection of two or more antibodies binding to saidtwo or more different tumor-associated antigens or to parts thereof ordetection of two or more cytotoxic or T helper lymphocytes specific forsaid two or more different tumor-associated antigens. In a furtherembodiment, the biological sample isolated from the patient is comparedto a comparable normal biological sample.

In a further aspect, the invention relates to a method for determiningregression, course or onset of a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises monitoring a sample from apatient who has said disease or is suspected of falling ill with saiddisease, with respect to one or more parameters selected from the groupconsisting of (i) the amount of nucleic acid which codes for thetumor-associated antigen or of a part thereof, (ii) the amount of thetumor-associated antigen or a part thereof, (iii) the amount ofantibodies which bind to the tumor-associated antigen or to a partthereof, and (iv) the amount of cytolytic T cells or T helper cellswhich are specific for a complex between the tumor-associated antigen ora part thereof and an MHC molecule. The method preferably comprisesdetermining the parameter(s) in a first sample at a first point in timeand in a further sample at a second point in time and in which thecourse of the disease is determined by comparing the two samples. Inparticular embodiments, the disease is characterized by expression orabnormal expression of two or more different tumor-associated antigensand monitoring comprises monitoring (i) the amount of two or morenucleic acids which code for said two or more different tumor-associatedantigens or of parts thereof, and/or (ii) the amount of said two or moredifferent tumor-associated antigens or of parts thereof, and/or (iii)the amount of two or more antibodies which bind to said two or moredifferent tumor-associated antigens or to parts thereof, and/or (iv) theamount of two or more cytolytic T cells or of T helper cells which arespecific for complexes between said two or more differenttumor-associated antigens or of parts thereof and MHC molecules.

According to the invention, detection of a nucleic acid or of a partthereof or monitoring the amount of a nucleic acid or of a part thereofmay be carried out using a polynucleotide probe which hybridizesspecifically to said nucleic acid or said part thereof or may be carriedout by selective amplification of said nucleic acid or said partthereof. In one embodiment, the polynucleotide probe comprises asequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguousnucleotides of said nucleic acid.

In particular embodiments, the tumor-associated antigen to be detectedor the part thereof is present intracellularly or on the cell surface.According to the invention, detection of a tumor-associated antigen orof a part thereof or monitoring the amount of a tumor-associated antigenor of a part thereof may be carried out using an antibody bindingspecifically to said tumor-associated antigen or said part thereof.

In further embodiments, the tumor-associated antigen to be detected orthe part thereof is present in a complex with an MHC molecule, inparticular an HLA molecule.

According to the invention, detection of an antibody or monitoring theamount of antibodies may be carried cut using a protein or peptidebinding specifically to said antibody.

According to the invention, detection of cytolytic T cells or of Thelper cells or monitoring the amount of cytolytic T cells or of Thelper cells which are specific for complexes between an antigen or apart thereof and MHC molecules may be carried out using a cellpresenting the complex between said antigen or said part thereof and anMHC molecule.

The polynucleotide probe, the antibody, the protein or peptide or thecell, which is used for detection or monitoring, is preferably labeledin a detectable manner. In particular embodiments, the detectable markeris a radioactive marker or an enzymic marker. T lymphocytes mayadditionally be detected by detecting their proliferation, theircytokine production, and their cytotoxic activity triggered by specificstimulation with the complex of MHC and tumor-associated antigen orparts thereof. T lymphocytes may also be detected via a recombinant MHCmolecule or else a complex of two or more MHC molecules which are loadedwith the particular immunogenic fragment of one or more of thetumor-associated antigens and which can identify the specific Tlymphocytes by contacting the specific T cell receptor.

In a further aspect, the invention relates to a method of treating,diagnosing or monitoring a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises administering an antibody whichbinds to said tumor-associated antigen or to a part thereof and which iscoupled to a therapeutic or diagnostic agent. The antibody may be amonoclonal antibody. In further embodiments, the antibody is a chimericor humanized antibody or a fragment of a natural antibody.

The invention also relates to a method of treating a patient having adisease characterized by expression or abnormal expression of atumor-associated antigen identified according to the invention, whichmethod comprises (i) removing a sample containing immunoreactive cellsfrom said patient, (ii) contacting said sample with a host cellexpressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic T cells against saidtumor-associated antigen or a part thereof, and (iii) introducing thecytolytic T cells into the patient in an amount suitable for lysingcells expressing the tumor-associated antigen or a part thereof. Theinvention likewise relates to cloning the T cell receptor of cytolytic Tcells against the tumor-associated antigen. Said receptor may betransferred to other T cells which thus receive the desired specificityand, as under (iii), may be introduced into the patient.

In one embodiment, the host cell endogenously expresses an HLA molecule.In a further embodiment, the host cell recombinantly expresses an HLAmolecule and/or the tumor-associated antigen or the part thereof. Thehost cell is preferably nonproliferative. In a preferred embodiment, thehost cell is an antigen-presenting cell, in particular a dendritic cell,a monocyte or a macrophage.

In a further aspect, the invention relates to a method of treating apatient having a disease characterized by expression or abnormalexpression of a tumor-associated antigen, which method comprises (i)identifying a nucleic acid which codes for a tumor-associated antigenidentified according to the invention and which is expressed by cellsassociated with said disease, (ii) transfecting a host cell with saidnucleic acid or a part thereof, (iii) culturing the transfected hostcell for expression of said nucleic acid (this is not obligatory when ahigh rate of transfection is obtained), and (iv) introducing the hostcells or an extract thereof into the patient in an amount suitable forincreasing the immune response to the patient's cells associated withthe disease. The method may further comprise identifying an MHC moleculepresenting the tumor-associated antigen or a part thereof, with the hostcell expressing the identified MHC molecule and presenting saidtumor-associated antigen or a part thereof. The immune response maycomprise a B cell response or a T cell response. Furthermore, a T cellresponse may comprise production of cytolytic T cells and/or T helpercells which are specific for the host cells presenting thetumor-associated antigen or a part thereof or specific for cells of thepatient which express said tumor-associated antigen or a part thereof.

The invention also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention, which method comprises(i) identifying cells from the patient which express abnormal amounts ofthe tumor-associated antigen, (ii) isolating a sample of said cells,(iii) culturing said cells, and (iv) introducing said cells into thepatient in an amount suitable for triggering an immune response to thecells.

Preferably, the host cells used according to the invention arenonproliferative or are rendered nonproliferative. A diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen is in particular cancer.

The present invention furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:2-5, 20-21, 31-33, 39, 54-57, 62, 63, 85-88, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c). Theinvention furthermore relates to a nucleic acid, which codes for aprotein or polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 7-13, 14-18, 23-24, 34-36, 58-61,64, 65, 89-100, a part or derivative thereof.

In a further aspect, the invention relates to promoter sequences ofnucleic acids of the invention. These sequences may be functionallylinked to another gene, preferably in an expression vector, and thusensure selective expression of said gene in appropriate cells.

In a further aspect, the invention relates to a recombinant nucleic acidmolecule, in particular DNA or RNA molecule, which comprises a nucleicacid of the invention.

The invention also relates to host cells which contain a nucleic acid ofthe invention or a recombinant nucleic acid molecule comprising anucleic acid of the invention.

The host cell may also comprise a nucleic acid coding for a HLAmolecule. In one embodiment, the host cell endogenously expresses theHLA molecule. In a further embodiment, the host cell recombinantlyexpresses the HLA molecule and/or the nucleic acid of the invention or apart thereof. Preferably, the host cell is nonproliferative. In apreferred embodiment, the host cell is an antigen-presenting cell, inparticular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, the invention relates to oligonucleotides whichhybridize with a nucleic acid identified according to the invention andwhich may be used as genetic probes or as “antisense” molecules. Nucleicacid molecules in the form of oligonucleotide primers or competentsamples, which hybridize with a nucleic acid identified according to theinvention or parts thereof, may be used for finding nucleic acids whichare homologous to said nucleic acid identified according to theinvention. PCR amplification, Southern and Northern hybridization may beemployed for finding homologous nucleic acids. Hybridization may becarried out under low stringency, more preferably under mediumstringency and most preferably under high stringency conditions. Theterm “stringent conditions” according to the invention refers toconditions which allow specific hybridization between polynucleotides.

In a further aspect, the invention relates to a protein or polypeptidewhich is encoded by a nucleic acid selected from the group consisting of(a) a nucleic acid which comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 2-5, 20-21, 31-33, 39, 54-57, 62,63, 85-88, a part or derivative thereof, (b) a nucleic acid whichhybridizes with the nucleic acid of (a) under stringent conditions, (c)a nucleic acid which is degenerate with respect to the nucleic acid of(a) or (b), and (d) a nucleic acid which is complementary to the nucleicacid of (a), (b) or (c). In a preferred embodiment, the inventionrelates to a protein or polypeptide which comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 7-13, 14-18,23-24, 34-36, 58-61, 64, 65, 89-100, a part or derivative thereof

In a further aspect, the invention relates to an immunogenic fragment ofa tumor-associated antigen identified according to the invention. Saidfragment preferably binds to a human HLA receptor or to a humanantibody. A fragment of the invention preferably comprises a sequence ofat least 6, in particular at least 8, at least 10, at least 12, at least15, at least 20, at least 30 or at least 50, amino acids.

In a further aspect, the invention relates to an agent which binds to atumor-associated antigen identified according to the invention or to apart thereof. In a preferred embodiment, the agent is an antibody. Infurther embodiments, the antibody is a chimeric, a humanized antibody oran antibody produced by combinatory techniques or is a fragment of anantibody. Furthermore, the invention relates to an antibody which bindsselectively to a complex of (i) a tumor-associated antigen identifiedaccording to the invention or a part thereof and (ii) an MHC molecule towhich said tumor-associated antigen identified according to theinvention or said part thereof binds, with said antibody not binding to(i) or (ii) alone. An antibody of the invention may be a monoclonalantibody. In further embodiments, the antibody is a chimeric orhumanized antibody or a fragment of a natural antibody.

The invention furthermore relates to a conjugate between an agent of theinvention which binds to a tumor-associated antigen identified accordingto the invention or to a part thereof or an antibody of the inventionand a therapeutic or diagnostic agent. In one embodiment, thetherapeutic or diagnostic agent is a toxin.

In a further aspect, the invention relates to a kit for detectingexpression or abnormal expression of a tumor-associated antigenidentified according to the invention, which kit comprises agents fordetection (i) of the nucleic acid which codes for the tumor-associatedantigen or of a part thereof, (ii) of the tumor-associated antigen or ofa part thereof, (iii) of antibodies which bind to the tumor-associatedantigen or to a part thereof, and/or (iv) of T cells which are specificfor a complex between the tumor-associated antigen or a part thereof andan MHC molecule. In one embodiment, the agents for detection of thenucleic acid or the part thereof are nucleic acid molecules forselective amplification of said nucleic acid, which comprise, inparticular a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of said nucleic acid.

According to the invention, genes are described which are expressed intumor cells selectively or aberrantly and which are tumor-associatedantigens.

According to the invention, these genes or their derivatives arepreferred target structures for therapeutic approaches. Conceptionally,said therapeutic approaches may aim at inhibiting the activity of theselectively expressed tumor-associated genetic product. This is useful,if said aberrant respective selective expression is functionallyimportant in tumor pathogenicity and if its ligation is accompanied byselective damage of the corresponding cells. Other therapeutic conceptscontemplate tumor-associated antigens as labels which recruit effectormechanisms having cell-damaging potential selectively to tumor cells.Here, the function of the target molecule itself and its role in tumordevelopment are totally irrelevant.

“Derivative” of a nucleic acid means according to the invention thatsingle or multiple nucleotide substitutions, deletions and/or additionsare present in said nucleic acid. Furthermore, the term “derivative”also comprises chemical derivatization of a nucleic acid on a nucleotidebase, on the sugar or on the phosphate. The term “derivative” alsocomprises nucleic acids which contain nucleotides and nucleotide analogsnot occurring naturally.

According to the invention, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the invention genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

According to the invention, complementary nucleic acids have at least40%, in particular at least 50%, at least 60%, at least 70%, at least80%, at least 90% and preferably at least 95%, at least 98 or at least99%, identical nucleotides.

Nucleic acids coding for tumor-associated antigens may, according to theinvention, be present alone or in combination with other nucleic acids,in particular heterologous nucleic acids. In preferred embodiments, anucleic acid is functionally linked to expression control sequences orregulatory sequences which may be homologous or heterologous withrespect to said nucleic acid. A coding sequence and a regulatorysequence are “functionally” linked to one another, if they arecovalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the invention promoters, enhancers and othercontrol elements which regulate expression of a gene. In particularembodiments of the invention, the expression control sequences can beregulated. The exact structure of regulatory sequences may vary as afunction of the species or cell type, but generally comprises 5′untranscribed and 5′ untranslated sequences which are involved ininitiation of transcription and translation, respectively, such as TATAbox, capping sequence, CAAT sequence, and the like. More specifically,5′ untranscribed regulatory sequences comprise a promoter region whichincludes a promoter sequence for transcriptional control of thefunctionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

Thus, on the one hand, the tumor-associated antigens illustrated hereinmay be combined with any expression control sequences and promoters. Onthe other hand, however, the promoters of the tumor-associated geneticproducts illustrated herein may, according to the invention, be combinedwith any other genes. This allows the selective activity of thesepromoters to be utilized.

According to the invention, a nucleic acid may furthermore be present incombination with another nucleic acid which codes for a polypeptidecontrolling secretion of the protein or polypeptide encoded by saidnucleic acid from a host cell. According to the invention, a nucleicacid may also be present in combination with another nucleic acid whichcodes for a polypeptide causing the encoded protein or polypeptide to beanchored on the cell membrane of the host cell or compartmentalized intoparticular organelles of said cell.

In a preferred embodiment, a recombinant DNA molecule is according tothe invention a vector, where appropriate with a promoter, whichcontrols expression of a nucleic acid, for example a nucleic acid codingfor a tumor-associated antigen of the invention. The term “vector” isused here in its most general meaning and comprises any intermediaryvehicle for a nucleic acid which enables said nucleic acid, for example,to be introduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. An intermediaryvehicle may be adapted, for example, to the use in electroporation, inbombardment with microprojectiles, in liposomal administration, in thetransfer with the aid of agrobacteria or in insertion via DNA or RNAviruses. Vectors comprise plasmids, phagemids or viral genomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the invention may be used for transfection of host cells.Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNAmay be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application. According to the invention, theterm “host cell” relates to any cell which can be transformed ortransfected with an exogenous nucleic acid. The term “host cells”comprises according to the invention prokaryotic (e.g. E. coli) oreukaryotic cells (e.g. dendritic cells, B cells, CHO cells, COS cells,K562 cells, yeast cells and insect cells). Particular preference isgiven to mammalian cells such as cells from humans, mice, hamsters,pigs, goats, primates. The cells may be derived from a multiplicity oftissue types and comprise primary cells and cell lines. Specificexamples comprise keratinocytes, peripheral blood leukocytes, stem cellsof the bone marrow and embryonic stem cells. In further embodiments, thehost cell is an antigen-presenting cell, in particular a dendritic cell,monocyte or a macrophage. A nucleic acid may be present in the host cellin the form of a single cop or of two or more copies and, in oneembodiment is expressed in the host cell.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selective markersuch as a gene imparting resistance to G418 (and thus enabling stablytransfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the invention in which an HLA molecule presents atumor-associated antigen or a part thereof, an expression vector mayalso comprise a nucleic acid sequence coding for said HLA molecule. Thenucleic acid sequence coding for the HLA molecule may be present on thesame expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter, case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe HLA molecule, both nucleic acids coding therefor are transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the HLA molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The invention also comprises kits for amplification of a nucleic acidcoding for a tumor-associated antigen. Such kits comprise, for example,a pair of amplification primers which hybridize to the nucleic acidcoding for the tumor-associated antigen. The primers preferably comprisea sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguousnucleotides of the nucleic acid and are nonoverlapping, in order toavoid the formation of primer dimers. One of the primers will hybridizeto one strand of the nucleic acid coding for the tumor-associatedantigen, and the other primer will hybridize to the complementary strandin an arrangement which allows amplification of the nucleic acid codingfor the tumor-associated antigen.

“Antisense” molecules or “antisense” nucleic acids may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the invention to an oligonucleotide which is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide or modifiedoligodeoxyribonucleotide and which hybridizes under physiologicalconditions to DNA comprising a particular gene or to mRNA of said gene,thereby inhibiting transcription of said gene and/or translation of saidmRNA. According to the invention, the “antisense molecule” alsocomprises a construct which contains a nucleic acid or a part thereof inreverse orientation with respect to its natural promoter. An antisensetranscript of a nucleic acid or of a part thereof may form a duplex withthe naturally occurring mRNA specifying the enzyme and thus preventaccumulation of or translation of the mRNA into the active enzyme,Another possibility is the use of ribozymes for inactivating a nucleicacid. Antisense oligonucleotides preferred according to the inventionhave a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of the target nucleic acid and preferably arefully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′ untranslated regionor mRNA splicing site.

In one embodiment, an oligonucleotide of the invention consists ofribonucleotides, deoxyribonucleotides or a combination thereof, with the5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the invention is a“modified” oligonucleotide. Here, the oligonucleotide may be modified invery different ways, without impairing its ability to bind its target,in order to increase, for example, its stability or therapeuticefficacy. According to the invention, the term “modifiedoligonucleotide” means an oligonucleotide in which (i) at least two ofits nucleotides are linked to one another by a synthetic internucleosidebond (i.e. an internucleoside bond which is not a phosphodiester bond)and/or (ii) a chemical group which is usually not found in nucleic acidsis covalently linked to the oligonucleotide. Preferred syntheticinternucleoside bonds are phosphorothioates, alkyl phosphonates,phosphorodithioates, phosphate esters, alkyl phosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, arboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

Preferably, the proteins and polypeptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpolypeptide” mean that the protein or polypeptide has been separatedfrom its natural environment. An isolated protein or polypeptide may bein an essentially purified state. The term “essentially purified” meansthat the protein or polypeptide is essentially free of other substanceswith which it is associated in nature or in vivo.

Such proteins and polypeptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and polypeptides described according to theinvention may be isolated from biological samples such as tissue or cellhomogenates and may also be expressed recombinantly in a multiplicity ofpro- or eukaryotic expression systems.

For the purposes of the present invention, “derivatives” of a protein orpolypeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible Amino acid deletion variants are characterized by theremoval of one or more amino acids from the sequence Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpolypeptides. Preference is given to replacing amino acids with otherones having similar properties such as hydrophobicity, hydrophilicity,electronegativity, volume of the side chain and the like (conservativesubstitution). Conservative substitutions, for example, relate to theexchange of one amino acid with another amino acid listed below in thesame group as the amino acid to be substituted:

1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly)

2. negatively charged residues and their amides: Asn, Asp, Glu, Gln

3. positively charged residues: His, Arg, Lys

4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val, (Cys)

5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residuesare shown in brackets. Gly is the only residue without a side chain andthus imparts flexibility to the chain. Pro has an unusual geometry whichgreatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. Techniques for introducing substitution mutations atpredetermined sites into DNA which has a known or partially knownsequence are well known and comprise M13 mutagenesis, for example. Themanipulation of DNA sequences for preparing proteins havingsubstitutions, insertions or deletions, is described in detail inSambrook et al. (1989), for example.

According to the invention, “derivatives” of proteins or polypeptidesalso comprise single or multiple substitutions, deletions and/oradditions of any molecules associated with the enzyme, such ascarbohydrates, lipids and/or proteins or polypeptides. The term“derivative” also extends to all functional chemical equivalents of saidproteins or polypeptides.

According to the invention, a part or fragment of a tumor-associatedantigen has a functional property of the polypeptide from which it hasbeen derived. Such functional properties comprise the interaction withantibodies, the interaction with other polypeptides or proteins, theselective binding of nucleic acids and an enzymatic activity. Aparticular property is the ability to form a complex with HLA and, whereappropriate, generate an immune response. This immune response may bebased on stimulating cytotoxic or T helper cells. A part or fragment ofa tumor-associated antigen of the invention preferably comprises asequence of at least 6, in particular at least 8, at least 10, at least12, at least 15, at least 20, at least 30 or at least 50, consecutiveamino acids of the tumor-associated antigen.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the invention to the part of the nucleicacid, which codes at least for the tumor-associated antigen and/or for apart or a fragment of said tumor-associated antigen, as defined above.

The isolation and identification of genes coding for tumor-associatedantigens also make possible the diagnosis of a disease characterized byexpression of one or more tumor-associated antigens. These methodscomprise determining one or more nucleic acids which code for atumor-associated antigen and/or determining the encoded tumor-associatedantigens and/or peptides derived therefrom. The nucleic acids may bedetermined in the conventional manner, including by polymerase chainreaction or hybridization with a labeled probe. Tumor-associatedantigens or peptides derived therefrom may be determined by screeningpatient antisera with respect to recognizing the antigen and/or thepeptides. They may also be determined by screening T cells of thepatient for specificities for the corresponding tumor-associatedantigen.

The present invention also enables proteins binding to tumor-associatedantigens described herein to be isolated, including antibodies andcellular binding partners of said tumor-associated antigens.

According to the invention, particular embodiments ought to involveproviding “dominant negative” polypeptides derived from tumor-associatedantigens. A dominant negative polypeptide is an inactive protein variantwhich, by way of interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery or whichcompetes with the active protein, thereby reducing the effect of saidactive protein. For example, a dominant negative receptor which binds toa ligand but does not generate any signal as response to binding to theligand can reduce the biological effect of said ligand. Similarly, adominant negative catalytically inactive kinase which usually interactswith target proteins but does not phosphorylate said target proteins mayreduce phosphorylation of said target proteins as response to a cellularsignal. Similarly, a dominant negative transcription factor which bindsto a promoter site in the control region of a gene but does not increasetranscription of said gene may reduce the effect of a normaltranscription factor by occupying promoter binding sites, withoutincreasing transcription.

The result of expression of a dominant negative polypeptide in a cell isa reduction in the function of active proteins. The skilled worker mayprepare dominant negative variants of a protein, for example, byconventional mutagenesis methods and by evaluating the dominant negativeeffect of the variant polypeptide.

The invention also comprises substances such as polypeptides which bindto tumor-associated antigens. Such binding substances may be used, forexample, in screening assays for detecting tumor-associated antigens andcomplexes of tumor-associated antigens with their binding partners andin a purification of said tumor-associated antigens and of complexesthereof with their binding partners. Such substances may also be usedfor inhibiting the activity of tumor-associated antigens, for example bybinding to such antigens.

The invention therefore comprises binding substances such as, forexample, antibodies or antibody fragments, which are capable ofselectively binding to tumor associated antigens. Antibodies comprisepolyclonal and monoclonal antibodies which are produced in theconventional manner.

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7^(th) Edition, Blackwell Scientific Publications, Oxford), The pFc′ andFc regions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

WO 92/04381 for example, describes production and use of humanizedmurine RSV antibodies in which at least part of the murine FR regionshave been replaced with FR regions of a human origin. Antibodies of thiskind, including fragments of intact antibodies with antigen-bindingcapability, are often referred to as “chimeric” antibodies.

The invention also provides F(ab′)₂, Fab, Fv, and Fd fragments ofantibodies, chimeric antibodies, in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, chimeric F(ab′)₂-fragmentantibodies in which the FR. and/or CDR1 and/or CDR2 and/or lightchain-CDR3 regions have been replaced with homologous human or nonhumansequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, and chimeric Fd-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced with homologous human or nonhuman sequences. The invention alsocomprises “single-chain” antibodies.

The invention also comprises polypeptides which bind specifically totumor-associated antigens. Polypeptide binding substances of this kindmay be provided, for example, by degenerate peptide libraries which maybe prepared simply in solution in an immobilized form or asphage-display libraries. It is likewise possible to preparecombinatorial libraries of peptides with one or more amino acids.Libraries of peptoids and nonpeptidic synthetic residues may also beprepared.

Phage display may be particularly effective in identifying bindingpeptides of the invention. In this connection, for example, a phagelibrary is prepared (using, for example, the M13, fd or lambda phages)which presents inserts of from 4 to about 80 amino acid residues inlength. Phages are then selected which carry inserts which bind to thetumor-associated antigen. This process may be repeated via two or morecycles of a reselection of phages binding to the tumor-associatedantigen. Repeated rounds result in a concentration of phages carryingparticular sequences. An analysis of DNA sequences may be carried out inorder to identify the sequences of the expressed polypeptides. Thesmallest linear portion of the sequence binding to the tumor-associatedantigen may be determined. The “two-hybrid system” of yeast may also beused for identifying polypeptides which bind to a tumor-associatedantigen. Tumor-associated antigens described according to the inventionor fragments thereof may be used for screening peptide libraries,including phage-display libraries, in order to identify and selectpeptide binding partners of the tumor-associated antigens. Suchmolecules may be used, for example, for screening assays, purificationprotocols, for interference with the function of the tumor-associatedantigen and for other purposes known to the skilled worker.

The antibodies described above and other binding molecules may be used,for example, for identifying tissue which expresses a tumor-associatedantigen. Antibodies may also be coupled to specific diagnosticsubstances for displaying cells and tissues expressing tumor-associatedantigens. They may also be coupled to therapeutically useful substances.Diagnostic substances comprise, in a nonlimiting manner, barium sulfate,iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate,meglumine diatrizoate, metrizamide, sodium tyropanoate and radiodiagnostic, including positron emitters such as fluorine-18 andcarbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131and indium-111, nuclides for nuclear magnetic resonance, such asfluorine and gadolinium. According to the invention, the term“therapeutically useful substance” means any therapeutic molecule which,as desired, is selectively guided to a cell which expresses one or moretumor-associated antigens, including anticancer agents, radioactiveiodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc.anticancer agents comprise, for example, aminoglutethimide,azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclpsporine, cytarabidine, dacarbazine,dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil,interferon-α, lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HC1, thioguanine, vinblastine sulfate and vincristinesulfate. Other anticancer agents are described, for example, in Goodmanand Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition,1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such aspokeweed antiviral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxinresidues may also be high energy-emitting radionuclides such ascobalt-60.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

According to the invention, the term “disease” refers to anypathological state in which tumor-associated antigens are expressed orabnormally expressed. “Abnormal expression” means according to theinvention that expression is altered, preferably increased, compared tothe state in a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50% orat least 100%. In one embodiment, the tumor-associated antigen isexpressed only in tissue of a diseased individual, while expression in ahealthy individual is repressed. One example of such a disease iscancer, in particular seminomas, melanomas, teratomas, gliomas,colorectal cancer, breast cancer, prostate cancer, cancer of the uterus,ovarian cancer and lung cancer.

According to the invention, a biological sample may be a tissue sampleand/or a cellular sample and may be obtained in the conventional mannersuch as by tissue biopsy, including punch biopsy, and by taking blood,bronchial aspirate, urine, feces or other body fluids, for use in thevarious methods described herein.

According to the invention, the term “immunoreactive cell” means a cellwhich can mature into an immune cell (such as B cell, T helper cell, orcytolytic T cell) with suitable stimulation Immunoreactive cellscomprise OD34⁺ hematopoietic stem cells, immature and mature T cells andimmature and mature B cells. If production of cytolytic or T helpercells recognizing a tumor-associated antigen is desired, theimmunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic T lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor detecting specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).Soluble MHC class I molecules are folded in vitro in the presence of β₂microglobulin and a peptide antigen binding to said class I molecule.The MHC/peptide complexes are purified and then labeled with biotin.Tetramers are formed by mixing the biotinylated peptide-MHC complexeswith labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1.Tetramers are then contacted with cytotoxic T lymphocytes such asperipheral blood or lymph nodes. The tetramers bind to cytotoxic Tlymphocytes which recognize the peptide antigen/MHC class I complex.Cells which are bound to the tetramers may be sorted byfluorescence-controlled cell sorting to isolate reactive cytotoxic Tlymphocytes. The isolated cytotoxic T lymphocytes may then be propagatedin vitro.

In a therapeutic method referred to as adoptive transfer (Greenberg, J.Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynchet al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell59:603-614, 1989), cells presenting the desired complex (e.g. dendriticcells) are combined with cytotoxic T lymphocytes of the patient to betreated, resulting in a propagation of specific cytotoxic T lymphocytes.The propagated cytotoxic T lymphocytes are then administered to apatient having a cellular anomaly characterized by particular abnormalcells presenting the specific complex. The cytotoxic T lymphocytes thenlyse the abnormal cells, thereby achieving a desired therapeutic effect.

Often, of the T cell repertoire of a patient, only T cells with lowaffinity for a specific complex of this kind can be propagated, sincethose with high affinity have been extinguished due to development oftolerance. An alternative here may be a transfer of the T cell receptoritself. For this too, cells presenting the desired complex (e.g.dendritic cells) are combined with cytotoxic T lymphocytes of healthyindividuals. This results in propagation of specific cytotoxic Tlymphocytes with high affinity if the donor had no previous contact withthe specific complex. The high affinity T cell receptor of thesepropagated specific T lymphocytes is cloned and can be transduced viagene transfer, for example using retroviral vectors, into T cells ofother patients, as desired. Adoptive transfer is then carried out usingthese genetically altered T lymphocytes (Stanislawski et al., NatImmunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61, 2001).

The therapeutic aspects above start out from the fact that at least someof the abnormal cells of the patient present a complex of atumor-associated antigen and an HLA molecule. Such cells may beidentified in a manner known per se. As soon as cells presenting thecomplex have been identified, they may be combined with a sample fromthe patient, which contains cytotoxic T lymphocytes. If the cytotoxic Tlymphocytes lyse the cells presenting the complex, it can be assumedthat a tumor-associated antigen is presented.

Adoptive transfer is not the only form of therapy which can be appliedaccording to the invention. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting HLA molecule). Various cell types may be used. Furthermore,it is possible to use vectors which carry one or both of the genes ofinterest. Particular preference is given to viral or bacterial vectors.For example, nucleic acids coding for a tumor-associated antigen or fora part thereof may be functionally linked to promoter and enhancersequences which control expression of said tumor-associated antigen or afragment thereof in particular tissues or cell types. The nucleic acidmay be incorporated into an expression vector. Expression vectors may benonmodified extrachromosomal nucleic acids, plasmids or viral genomesinto which exogenous nucleic acids may be inserted. Nucleic acids codingfor a tumor-associated antigen may also be inserted into a retroviralgenome, thereby enabling the nucleic acid to be integrated into thegenome of the target tissue or target cell. In these systems, amicroorganism such as vaccinia virus, pox virus, Herpes simplex virus,retrovirus or adenovirus carries the gene of interest and de facto“infects” host cells. Another preferred form is the introduction of thetumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the HLA molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toHLA molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp at al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen- to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyat al., Proc Natl Aced Sci USA 98:3299-303, 2001). It may also becarried out in combination with reagents which facilitate uptake intodendritic cells. In vivo preferred tumor-associated antigens comprisethose which react with allogenic cancer antisera or with T cells of manycancer patients. Of particular interest, however, are those againstwhich no spontaneous immune responses pre-exist. Evidently, it ispossible to induce against these immune responses which can lyse tumors(Keogh at al., J. Immunol. 167:787-96, 2001; Appella at al., Biomed PeptProteins Nucleic Acids 1:177-84, 1995; Wentworth et al., Mol Immunol.32:603-12, 1995).

The pharmaceutical compositions described according to the invention mayalso be used as vaccines for immunization. According to the invention,the terms “immunization” or “vaccination” mean an increase in oractivation of an immune response to an antigen. It is possible to useanimal models for testing an immunizing effect on cancer by using atumor-associated antigen or a nucleic acid coding therefor. For example,human cancer cells may be introduced into a mouse to generate a tumor,and one or more nucleic acids coding for tumor-associated antigens maybe administered. The effect on the cancer cells (for example reductionin tumor size) may be measured as a measure for the effectiveness of animmunization by the nucleic acid.

As part of the composition for an immunization, one or moretumor-associated antigens or stimulating fragments thereof areadministered together with one or more adjuvants for inducing an immuneresponse or for increasing an immune response. An adjuvant is asubstance which is incorporated into the antigen or administeredtogether with the latter and which enhances the immune response.Adjuvants may enhance the immune response by providing an antigenreservoir (extracellularly or in macrophages), activating macrophagesand stimulating particular lymphocytes. Adjuvants are known and comprisein a nonlimiting way monophosphoryl lipid A (MPL, SmithKline Beecham),saponin such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells 7:178-186,1997), incomplete Freund's adjuvant, complete Freund's adjuvant, vitaminE, montanide, alum, CpG oligonucleotides (cf. Kreig at al., Nature374:546-9, 1995) and various water-in-oil emulsions prepared frombiologically degradable oils such as squalene and/or tocopherol.Preferably, the peptides are administered in a mixture with DQS21/MPL.The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and in particular about 1:1. For administration tohumans, a vaccine formulation typically contains DQS21 and MPL in arange from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids. Examples of such costimulating molecules are B7-1 and B7-2 (CD80and CD86, respectively) which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cells. Thisinteraction provides a costimulation (signal 2) for anantigen/MHC/TCR-stimulated (signal 1) T cell, thereby enhancingpropagation of said T cell and the effector function. B7 also interactswith CTLA4 (CD152) on T cells, and, studies involving CTLA4 and B7ligands demonstrate that B7-CTLA4 interaction can enhance antitumorimmunity and CTL propagation (Zheng, P. et al., Proc. Natl. Acad. Sci.USA 95(11):6284-6289 (1998)).

B7 is typically not expressed on tumor cells so that these are noeffective antigen-presenting cells (APCs) for T cells. Induction of B7expression would enable rumor cells to stimulate more effectivelypropagation of cytotoxic T lymphocytes and an effector function.Costimulation by a combination of B7/IL-6/IL-12 revealed induction ofIFN-gamma and Th1-cytokine profile in a T cell population, resulting infurther enhanced T cell activity (Gajewski et al., J. Inmunol.154:5637-5648 (1995)).

A complete activation of cytotoxic T lymphocytes and a complete effectorfunction require an involvement of T helper cells via interactionbetween the CD40 ligand on said T helper cells and the CD40 moleculeexpressed by dendritic cells (Ridge et al., Nature 393:474 (1998),Bennett et al., Nature 393:478 (1998), Schönberger et al., Nature393:480 (1998)). The mechanism of this costimulating signal probablyrelates to the increase in B7 production and associated IL-6/IL-12production by said dendritic cells (antigen-presenting cells).CD40-CD40L interaction thus complements the interaction of signal I(antigen/MHC-TCR) and signal 2 (B7-CD28).

The use of anti-CD40 antibodies for stimulating dendritic cells would beexpected to directly enhance a response to tumor antigens which areusually outside the range of an inflammatory response or which arepresented by nonprofessional antigen-presenting cells (tumor cells). Inthese situations, T helper and B7-costimulating signals are notprovided. This mechanism could be used in connection with therapiesbased on antigen-pulsed dendritic cells or in situations in which Thelper epitopes have not been defined in known TRA precursors.

The invention also provides for administration of nucleic acids,polypeptides or peptides. Polypeptides and peptides may be administeredin a manner known per se. In one embodiment, nucleic acids areadministered by ex vivo methods, i.e, by removing cells from a patient,genetic modification of said cells in order to incorporate atumor-associated antigen and reintroduction of the altered cells intothe patient. This generally comprises introducing a functional copy of agene into the cells of a patient in vitro and reintroducing thegenetically altered cells into the patient. The functional copy of thegene is under the functional control of regulatory elements which allowthe gene to be expressed in the genetically altered cells. Transfectionand transduction methods are known to the skilled worker. The inventionalso provides for administering nucleic acids in vivo by using vectorssuch as viruses and target-controlled liposomes.

In a preferred embodiment, a viral vector for administering a nucleicacid coding for a tumor-associated antigen is selected from the groupconsisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e, they areincapable of generating infectious particles).

Various methods may be used in order to introduce according to theinvention nucleic acids into cells in vitro or in vivo. Methods of thiskind comprise transfection of nucleic acid CaPO₄ precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the invention may be administered inpharmaceutically compatible preparations. Such preparations may usuallycontain pharmaceutically compatible concentrations of salts, buffersubstances, preservatives, carriers, supplementing immunity-enhancingsubstances such as adjuvants, CpG and cytokines and, where appropriate,other therapeutically active compounds.

The therapeutically active compounds of the invention may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lunch aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the invention are administered in effective amounts.An “effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of particular conditioncharacterized by expression of one or more tumor-associated antigens,the desired reaction relates to inhibition of the course of the disease.This comprises slowing down the progress of the disease and, inparticular, interrupting the progress of the disease. The desiredreaction in a treatment of a disease or of a condition may also be delayof the onset or a prevention of the onset of said disease or saidcondition.

An effective amount of a composition of the invention will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect,

The doses administered of the compositions of the invention may dependon various parameters such as the type of administration, the conditionof the patient, the desired period of administration, etc. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg, are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may be used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier. According to the invention, theterm “pharmaceutically compatible carrier” refers to one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the invention are usually such that no interaction occurs whichsubstantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, suspensions, syrups, elixir or in the form of an emulsion, forexample.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesherein, which are used only for illustration purposes and are not meantto be limiting, Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES Material and Methods

The terms “in silico”, “electronic” and “virtual cloning” refer solelyto the utilization of methods based on databases, which may also be usedto simulate laboratory experimental processes.

Unless expressly defined otherwise, all other terms and expressions areused so as to be understood by the skilled worker. The techniques andmethods mentioned are carried out in a manner known per se and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. All methods including the use of kitsand reagents are carried out according to the manufacturers'information.

Datamining-Based Strategy for Determining eCT (Electronically ClonedCancer/Testis Genes)

Two in silico strategies, namely GenBank keyword search and thecDNAxProfiler, were combined (FIG. 1). Utilizing the NCBI ENTREZ Searchand Retrieval System (available at NCBI website), a GenBank search wascarried out for candidate genes annotated as being specificallyexpressed in testicular tissue (Wheeler at al., Nucleic Acids Research28:10-14, 2000).

Carrying out queries with the keywords “testis-specific gene”,“sperm-specific gene”, “spermatogonia-specific gene”, candidate genes(GOI, genes of interest) were extracted from the databases. The searchwas restricted to part of the total information of these databases byusing the limits “homo sapiens”, for the organism, and “mRNA”, for thetype of molecule.

The list of the GOI found was curated by determining different names forthe same sequence and eliminating such redundancies.

All candidate genes obtained by the keyword search were in turn studiedwith respect to their tissue distribution by the “electronic Northern”(eNorthern) method. The eNorthern is based on aligning the sequence of aGOI with an EST (expressed sequence tag) database (Adams et al., Science252:1651, 1991) (BLAST search at NCBI website). The tissue origin ofeach EST which is found to be homologous to the GOI can be determinedand in this way the sum of all ESTs produces a preliminary assessment ofthe tissue distribution of the GOI. Further studies were carried outonly with those GOI which had no homologies to EST from nontesticularnormal tissues with the exception of placenta and fetal tissue. Thisevaluation also took into account that the public domain containswrongly annotated cDNA libraries (Scheurle et al., Cancer Res,60:4037-4043, 2000).

The second datamining method utilized was the cDNA xProfiler of the NCBICancer Genome Anatomy Project (available at NCI website) (Hillier etal., Genome Research 6:807-828, 1996; Pennisi, Science 276:1023-1024,1997). This allows pools of transcriptomes deposited in databases to berelated to one another by logical operators. We have defined a pool A towhich all expression libraries prepared from testis were assigned,excluding mixed libraries. All cDNA libraries prepared from normaltissues other than testis, ovary or fetal tissue were assigned to poolB. Generally, all cDNA libraries were utilized independently ofunderlying preparation methods, but only those with a size >1000 wereadmitted. Pool B was digitally subtracted from pool A by means of theBUT NOT operator. The set of GOI found in this manner was also subjectedto eNorthern studies and validated by a literature research.

This combined datamining includes all of the about 13000 full-lengthgenes in the public domain and predicts out of these genes a total of140 genes having potential testis-specific expression, Among the latterwere 25 previously known genes of the CT gene class, underlining theefficiency of our strategy.

All other genes were first evaluated in normal tissues by means ofspecific RT-PCR. All GOI which had proved to be expressed innontesticular normal tissues had to be regarded as false-positives andwere excluded from further studies. The remaining ones were studied in alarge panel of a wide variety of tumor tissues. The antigens depictedbelow proved here to be ectopically activated in tumor cells.

RNA Extraction, Preparation of Poly-d(T) Primed cDNA and RT-PCR Analysis

Total RNA was extracted from native tissue material by using guanidiumisothiocyanate as chaotropic agent (Chomczynski & Sacchi, Anal. Biochem.162:156-9, 1987). After extraction with acidic phenol and precipitationwith isopropanol, said RNA was dissolved in DEPC treated water.

First strand cDNA synthesis from 2-4 μg of total RNA was carried out ina 20 μl reaction mixture by means of Superscript II (Invitrogen),according to the manufacturer's information. The primer used was adT(18) oligonucleotide. Integrity and quality of the cDNA were checkedby amplification of p53 in a 30 cycle PCR (senseCGTGAGCGCTTCGAGATGTTCCG, (SEQ ID NO:52) antisenseCCTAACCAGCTGCCCAACTGTAG, (SEQ ID NO:53) hybridization temperature 67°C.).

An archive of first strand cDNA was prepared from a number of normaltissues and tumor entities. For expression studies, 0.5 μl of thesecDNAs was amplified in a 30 μl reaction mixture, using GOI-specificprimers (see below) and 1 U of HotStarTaq DNA polymerase (Oiagen). Eachreaction mixture contained 0.3 mM dNTPs, 0.3 μM of each primer and 3 μlof 10× reaction buffer. The primers were selected so as to be located intwo different exons, and elimination of the interference bycontaminating genomic DNA as the reason for false-positive results wasconfirmed by testing nonreverse-transcribed DNA as template. After 15minutes at 95° C. to activate the HotStarTaq DNA polymerase, 35 cyclesof PCR were carried out (1 min at 94° C., 1 min at the particularhybridization temperature, 2 min at 72° C. and final elongation at 72°C. for 6 min).

20 μl of this reaction were fractionated and analyzed on an ethidiumbromide-stained agarose gel.

The following primers were used for expression analysis of thecorresponding antigens at the hybridization temperature indicated.

LDH-C (67° C.) (SEQ ID NO: 25) sense TGCCGTAGGCATGGCTTGTGC, (SEQ ID NO:26) antisense CAACATCTGAGACACCATTCC TPTE (64° C.) (SEQ ID NO: 27) senseTGGATGTCACTCTCATCCTTG, (SEQ ID NO: 28) antisense CCATAGTTCCTGTTCTATCTGTSBP (63° C.) (SEQ ID NO: 42) sense TCTAGCACTGTCTCGATCAAG, (SEQ ID NO:43) antisense TGTCCTCTTGGTACATCTGAC MS4A12 (66°) (SEQ ID NO: 44) senseCTGTGTCAGCATCCAAGGAGC, (SEQ ID NO: 45) antisense TTCACCTTTGCCAGCATGTAGBRCO1 (60′C) (SEQ ID NO: 46) sense CTTGCTCTGAGTCATCAGATG, (SEQ ID NO:47) antisense CACAGAATATGAGCCATACAG TPX1 (65° C.) (SEQ ID NO: 78) senseTTTTGTCTATGGTGTAGGACC, (SEQ ID NO: 79) antisense GGAATGGCAATGATGTTACAG.

Preparation of Random Hexamer-Primed cDNA and Quantitative Real Time PCR

LDHC expression was quantified by means of real time PCR.

The principle of quantitative real time PCR using the ABI PRISM SequenceDetection System (PE Biosystems, USA) utilizes the 5′-3′ exonucleaseactivity of Taq DNA polymerase for direct and specific detection of PCRproducts via release of fluorescence reporter dyes. In addition to senseand antisense primers, the PCR employs a doubly fluorescently labeledprobe (TaqMan probe) which hybridizes to a sequence of the PCR product.The probe is labeled 5′ with a reporter dye (e.g. FAM) and 3′ with aquencher dye (e.g. TAMRA). If the probe is intact, the spatial proximityof reporter to quencher suppresses the emission of reporterfluorescence. If the probe hybridizes to the PCR product during the PCR,said probe is cleaved by the 5′-3′ exonuclease activity of Taq DNApolymerase and suppression of the reporter fluorescence is removed. Theincrease in reporter fluorescence as a consequence of the amplificationof the target, is measured after each PCR cycle and utilized forquantification. Expression of the target gene is quantified absolutelyor relative to expression of a control gene with constant expression inthe tissues to be studied. LDHC expression was calculated by means ofthe ΔΔ-C_(t) method (PE Biosystems, USA), after normalizing the samplesto 18s RNA as “housekeeping” gene. The reactions were carried out induplex mixtures and determined in duplicate. cDNA was synthesized usingthe High Capacity cDNA Archive Kit (PE Biosystems, USA) and hexamerprimers according to the manufacturer's information. In each case 5 μlof the diluted cDNA were used for the PCR in a total volume of 25 μl:sense primer (GGTGTCACTTCTGTGCCTTCCT) (SEQ ID NO:48) 300 nM; antisenseprimer (CGGCACCAGTTCCAACAATAG) (SEQ ID NO:49) 300 nM; TaqMan probe(CAAAGGTTCTCCAAATGT) (SEQ ID NO:50) 250 nM; sense primer 18s RNA 50 nM;antisense primer 18s RNA 50 nM; 18s RNA sample 250 nM; 12.5 μl TaqManUniversal PCR Master Mix; initial denaturation 95° C. (10 min); 95° C.(15 sec); 60° C. (1 min); 40 cycles. Due to amplification of a 128 bpproduct beyond the border of exon 1 and exon 2, all LDHC splice variantsdescribed were included in the quantification.

Cloning and Sequence Analysis

Full length genes and gene fragments were cloned by common methods. Thesequence was determined by amplifying corresponding antigens by means ofthe pfu proofreading polymerase (Stratagene). After completion of thePCR, adenosine was ligated by means of HotStarTaq DNA polymerase to theends of the amplicon in order to clone the fragments into the TOPO-TAvector according to the manufacturer's information. A commercial servicecarried out the sequencing. The sequences were analyzed by means ofcommon prediction programs and algorithms.

Example 1 Identification of LDH C as a New Tumor Antigen

LDH C (SEQ ID NO:1) and its translation product (SEQ ID NO:6) have beendescribed as testis-specific isoenzyme of the lactate dehydrogenasefamily. The sequence has been published in GenBank under accessionnumber NM_017448. The enzyme forms a homotetramer having a molecularweight of 140 kDa (Goldberg, E. et al., Contraception 64(2):93-8, 2001;Cooker et al., Biol. Reprod. 48(6):1309-19, 1993; Gupta, G. S., Crit.Rev. Biochem, Mol. Biol. 34(6):361-85, 1999).

RT-PCR studies for expression analysis using a primer pair(5′-TGCCGTAGGCATGGCTTGTGC-3′, (SEQ ID NO:25) and5′-CAACATCTGAGACACCATTCC-3′) (SEQ ID NO:26) which does not cross-amplifythe related and ubiquitously expressed isoenzymes LDH A and LDH B andwhich is based on the LDH C prototype sequence NM_017448 which haspreviously been described as being testis-specific, confirmed accordingto the invention the lack of expression in all normal tissues tested,but demonstrated that the stringent transcriptional repression of thisantigen in somatic cells has been removed in the case of tumors; cf.Table 1. As has been described classically for CT genes, LDH C isexpressed in a number of tumor entities.

TABLE 1 Expression of LDHC in tumors Tested Tissue in total Positive %Melanoma 16 7 44 Mammary carcinomas 20 7 35 Colorectal tumors 20 3 15Prostate carcinomas 8 3 38 Bronchial carcinomas 17 8 47 Kidney cellcarcinomas 7 4 57 Ovarian carcinomas 7 3 43 Thyroid carcinomas 4 1 25Cervical carcinomas 6 5 83 Melanoma cell lines 8 5 63 Bronchialcarcinoma cell lines 6 2 33

The expected size of the amplification product is 824 bp, using the PCRprimers mentioned above. According to the invention, however,amplification of multiple additional bands was observed in tumors, butnot in testis. Since this is indicative for the presence of alternativesplice variants, the complete open reading frame was amplified usingLDH-C-specific primers (5′-TAGCGCCTCAACTGTCGTTGG-3′, (SEQ ID NO:51) and5′-CAACATCTGAGACACCATTCC-3′) (SEQ ID NO:26) and independent full-lengthclones were sequenced. Alignments with the prototype ORF of the LDH Csequence described (SEQ ID NO:1) and the genomic sequence on chromosome11 confirm additional splice variants (SEQ ID NO:2-5). The alternativesplicing events result in the absence of exon 3 (SEQ ID NO:2), of thetwo exons 3 and 4 (SEQ ID NO:3), of the exons 3, 6 and 7 (SEQ ID NO:4)or of exon 7 (SEQ ID NO:5) (cf. FIG. 2).

These new splice variants are generated exclusively in tumors, but notin testis. Alternative splicing causes alterations in the reading frameand results in new possible ORFS encoding the amino acid sequencesdepicted in SEQ ID NO:7-13 (ORF for SEQ ID NO:7: nucleotide position59-214 of SEQ ID NO:2 and, respectively, SEQ ID NO:4 ORF for SEQ IDNO:8: nucleotide position 289-939 of SEQ ID NO:2; ORF for SEQ ID NO:9:nucleotide position 59-196 of SEQ ID NO:3; ORF for SEQ ID NO:10:nucleotide position 535-765 of SEQ ID NO:3; ORF for SEQ ID NO:11:nucleotide position 289-618 of SEQ ID NO:4; ORF for SEQ ID NO:12:nucleotide position 497-697 of SEQ ID NO:4; ORF for SEQ ID NO:13:nucleotide position 59-784 of SEQ ID NO:5) (FIG. 2, 3). Apart frompremature termination, utilization of alternative start codons is alsopossible so that the encoded proteins may be truncated both N-terminallyand C-terminally.

While SEQ ID NO:8 and SEQ ID NO:10 represent truncated portions of theprototype protein, the protein sequence of SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:12 and SEQ ID NO:13 are additionally altered andcontain only tumor-specific epitopes (printed in bold type in FIG. 3).Peptide regions which could result in tumor specific epitopes are asfollows (the strictly tumor-specific portion produced by frame shifts isunderlined):

SEQ ID NO: 14: GAVGMACAISILL KITVYLQTPE  (of SEQ ID NO: 7) SEQ ID NO:15: GAVGMACAISILLK WIF  (of SEQ ID NO: 9) SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWC IRCCEN  (of SEQ ID NO: 11) SEQ 7D NO: 17:MVGLLENMVILV GLYGIKEELFL (of SEQ ID NO: 12) SEQ ID NO: 18: EHWKNIHKQVIQRDYME  (of SEQ ID NO: 13)

These regions may potentially contain epitopes which can be recognizedon MHC I or MHC II molecules by T lymphocytes and which result in astrictly tumor-specific response.

Not all of the predicted proteins have the catalytic lactatedehydrogenase domain for NADH-dependent metabolization of pyruvate tolactate, which represents the last step of anaerobic glycolysis. Thisdomain would be required for the enzymatic function as lactatedehydrogenase (framed in FIG. 3). Further analyses, for example usingalgorithms such as TMpred and pSORT (Nakai & Kanehisa, 1992), predictdifferent subcellular localizations for the putative proteins.

According to the invention, the level of expression was quantified byreal time PCR using a specific primer-sample set. The amplicon ispresent in the junction between exon 1 and exon 2 and thus detects allvariants (SEQ ID NO:1-5). These studies too, do not detect anytranscripts in normal tissues except testis. They confirm significantlevels of expression in tumors (FIG. 4).

LDHC-specific polyclonal antibodies were produced according to theinvention by selecting a peptide from the extreme N-terminal regionMSTVKEQLIEKLIEDDENSQ (SEQ ID NO:80). LDHC-specific antibodies wereproduced in rabbits with the aid of this peptide. Subsequent studies onprotein expression confirmed selective LDHC expression in testis and invarious tumors. In addition, immunohistological studies in accordancewith the invention revealed a distinct colocalization of LDHC withcytochrome C oxidase in mitochondria. This indicates that LDHC plays animportant part in the respiratory chain of tumors.

Example 2 Identification of TPTE as a New Tumor Antigen

The sequences of the TPTE transcript (SEQ ID NO:19) and of itstranslation product (SEQ ID NO:22) have been published in GenBank underaccession number NM_013315 (Walker, S. M. at al., Biochem. J. 360(Pt2):277-83, 2001; Guipponi M. et al., Hum. Genet. 107(2):127-31, 2000;Chen H. et al., Hum. Genet. 105(5):399-409, 1999) TPTE has beendescribed as a gene coding for a possible transmembranetyrosinephosphatase, with testis-specific expression located in thepericentromeric region of chromosomes 21, 13, 15, 22 and Y (Chen, H. etal., Hum. Genet. 105:399-409, 1999). Alignment studies in accordancewith the invention additionally reveal homologous genomic sequences onchromosomes 3 and 7.

According to the invention, PCR primers (5′-TGGATGTCACTCTCATCCTTG-3′(SEQ ID NO:27) and 5′-CCATAGTTCCTGTTCTATCTG-3′) (SEQ ID NO:28) weregenerated based on the sequence of TPTE (SEQ ID NO:19) and used forRT-PCR analyses (95° 15 min; 94° 1 min; 63° 1 min; 72° 1 min; 35 cycles)in a number of human tissues. Expression in normal tissues was shown tobe limited to testis. As described for the other eCT, TPTE variants wereshown according to the invention to be ectopically activated in a numberof tumor tissues; cf. Table 2. According to the invention, further TPTEsplice variants were identified (SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57) which are expressed intesticular tissue and in tumors and which have frame shifts and thusaltered sequence regions (FIG. 5).

TABLE 2 Expression of TPTE in tumors Tested Tissue in total Positive %Melanoma 18 9 50 Mammary carcinomas 20 4 20 Colorectal tumors 20 0 0Prostate carcinomas 8 3 38 Bronchial carcinomas 23 9 39 Kidney cellcarcinomas 7 0 0 Ovarian carcinomas 7 2 29 Thyroid carcinomas 4 0 0Cervical carcinomas 6 1 17 Melanoma cell lines 8 4 50 Bronchialcarcinoma cell lines 6 2 33 Mammalian carcinoma cell lines 5 4 80

The TPTE genomic sequence consists of 24 axons (accession numberNT_029430). The transcript depicted in SEQ ID NO:19 contains all ofthese exons. The splice variant depicted in SEQ ID NO:20 is produced bysplicing out exon 7. The splice variant depicted in SEQ ID NO:21 showspartial incorporation of an intron downstream of exon 15. As thevariants SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57indicate, it is alternatively also possible to splice out exons 18, 19,20 and 21.

These alternative splicing events result in alterations of the encodedprotein, with the reading frame being retained in principle (FIG. 6).For example, the translation product encoded by the sequence depicted inSEQ ID NO:20 (SEQ ID NO:23) has a deletion of 13 amino acids incomparison to the sequence depicted in SEQ ID NO:22. The translationproduct encoded by the sequence depicted in SEQ ID NO:21 (SEQ ID NO:24)carries an additional insertion in the central region of the moleculeand thereby differs from the other variants by 14 amino acids.

The translation products of the variants SEQ ID NO:54, SEQ ID NO:55, SEQID NO:56, SEQ ID NO:57, namely the proteins SEQ ID NO:58, SEQ) ID NO:59,SEQ ID NO:60, SEQ ID NO:61, are likewise altered.

Analyses for predicting the functional domains reveal the presence of atyrosinephosphatase domain for SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:58, SEQ ID NO:60 but not for SEQ ID NO:59, SEQ ID NO:61. Forall variants, 3-4 transmembrane domains are predicted (FIG. 6).

Analysis of TPTE antigen expression, using specific antibodies,confirmed selective expression in testis and in a number of differenttumors. Colocalization studies moreover revealed that according to theinvention TPTE is located together with class I immunoglobulins on thecell surface of tumor cells. Previously, TPTE had been described only asa Golgi-associated protein. Owing to TPTE expression on the cell surfaceof tumor cells, this tumor antigen is suitable according to theinvention as an outstanding target for developing diagnostic andtherapeutic monoclonal antibodies. Owing to the predicted membranetopology of TPTE, the extracellulary exposed regions are particularlysuitable for this purpose according to the invention. According to theinvention, this comprises the peptides FTDSKLYIPLEYRS (SEQ ID NO:81) andFDIKLLRNIPRWT (SEQ ID NO: 82). In addition, TPTE was shown to promotethe migration of tumor cells. To this end, tumor cells which had beentransfected with TPTE under the control of a eukaryotic promoter andcontrol cells were studied in “Boyden chamber” migration experiments, asto whether they exhibit directed migration. TPTE-transfected cells herehad according to the invention markedly (3-fold) increased migration in4 independent experiments. These functional data indicate that TPTEplays an important part in the metastasizing of tumors. Thus, processeswhich inhibit according to the invention endogenous TPTE activity intumor cells, for example by using antisense RNA, different methods ofRNA interference (RNAi) by means of expression vectors or retroviruses,and by using small molecules, could result in reduced metastasizing andthus be very important therapeutically. A causal connection between theactivity of a phosphatase in tumors and increased migration andincreased formation of metastases was established recently for the PTENtyrosinephosphastase (Iijima and Devreotes Cell 109:599-610, 2002).

Example 3 Identification of TSBP as a New Tumor Antigen

The electronic cloning method employed according to the inventionproduced TSBP (SEQ ID NO:29) and the protein derived therefrom (SEQ IDNO:30). The gene has been described previously as beingtestis-specifically regulated (accession number NM_006781). The gene waspredicted to encode a basic protein and to be located on chromosome 6close to a sequence coding for an MHC complex (C6orf10) (Stammers M. etal., Immunogenetics 51(4-5):373-82, 2000). According to the invention,the previously described sequence was shown to be incorrect. Thesequence of the invention is substantially different from the knownsequence. According to the invention, 3 different splicing variants werecloned. The differences in the nucleotide sequences of the TSBP variantsfound according to the invention (SEQ ID NO:31, SEQ ID NO: 32, SEQ IDNO:33) to the known sequence (NM_006781, SEQ ID NO:29) are depicted inFIG. 7 (differences depicted in bold type). They result in frame shiftsso that the proteins encoded by the TSBP variants found according to theinvention (SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36) differsubstantially from the previously described protein (SEQ ID NO:30) (FIG.8).

It was confirmed according to the invention that this antigen isstrictly transcriptionally repressed in normal tissues (PCR primers5′-TCTAGCACTGTCTCGATCAAG-3′ (SEQ ID NO:42) and5′-TGTCCTCTTGGTACATCTGAC-3′) (SEQ ID NO:43). However, in 25 normaltissues studied, TSBP was expressed, apart from in testis, also innormal lymph node tissue. According to the invention, ectopic activationof TSBP in tumors was also detected, and it therefore qualifies as atumor marker or tumor-associated antigen (Table 3).

Although TSBP, expression is found in primary tumor tissue, it is notfound in permanent cell lines of corresponding tumor entities. Moreover,the gene is in the direct neighborhood of Notch 4 which is specificallyexpressed in arteries and involved in vascular morphogenesis. These aresignificant indications of this being a marker for specific endothelialcells. TSBP may therefore serve as a potential marker for tumorendothelia and for neovascular targeting.

Consequently, the TSBP promoter may be cloned to another genetic productwhose selective expression in lymph nodes is desired.

Analysis of TSBP antigen expression, using specific antibodies,confirmed the selective localization of the protein in testis and lymphnodes and also in melanomas and bronchial carcinomas. In addition,immunohistological studies using GFP-tagged TSBP revealed a distinctperinucleic accumulation.

TABLE 3 Expression of TSBP in tumors Tested Tissue in total Positive %Melanoma 12 2 16 Mammary carcinomas 15 0 — Colorectal tumors 15 0 —Prostate carcinomas 8 0 — Bronchial carcinomas 7 17 41 Kidney cellcarcinomas 7 0 — Ovarian carcinomas 7 0 — Thyroid carcinomas 4 0 —Cervical carcinomas 6 0 — Melanoma cell lines 8 0 — Bronchial carcinomacell lines 6 0 —

Example 4 Identification of MS4Al2 as a New Tumor Antigen

MS4Al2 (SEQ ID NO:37, accession number NM_017716) and its translationproduct (SEQ ID NO:38) have been described previously as members of amultigene family related to the B cell-specific antigen CD20, thehematopoietic cell-specific protein HTm4 and the β chain of the highaffinity IgE receptor. All family members are characterized by at leastfour potential transmembrane domains and both the C and the N-terminusare cytoplasmic (Liang Y. et al., Immunogenetics 53(5):357-68, 2001;Liang Y. & Tedder, Genomics 72(2):119-27, 2001). According to theinvention, RT-PCR studies on MS4Al2 were carried out. The primers wereselected based on the published MS4Al2 sequence (NM_017716) (sense:CTGTGTCAGCATCCAAGGAGC, (SEQ ID NO:44) antisense: TTCACCTTTGCCAGCATGTAG)(SEQ ID NO:45). In the tissues tested, expression was detected only intestis, colon (6/8) and colorectal carcinomas (colon-Ca's) (16/20) andin colonic metastases (12/15) (FIG. 9).

The high incidence in colonic metastases makes TSBP an attractivediagnostic and therapeutic target. According to the invention, thepredicted extracellular region comprising the protein sequenceGVAGQDYWAVLSGKG (SEQ ID NO:83) is particularly suitable for producingmonoclonal antibodies and small chemical inhibitors. According to theinvention, the intracellular localization of the MS4Al2 protein on thecell membrane was also confirmed by fluorescence superposition usingplasma membrane markers in confocal immunofluorescence.

TABLE 4 Expression of MS4Al2 in normal tissues and colorectal carcinomasand metastasis Ileum + Colon + Liver − Lung − Lymph nodes − Stomach −Spleen − Adrenal gland − Kidney − Esophagus − Ovary − Rectum + Testis +Thymus − Skin − Mamma − Pancreas − PBMC − PBMC act. − Prostate − Thyroid− Tube − Uterus − Cerebrum − Cerebellum − Colorectal tumors 16/20Colorectal tumors metastases 12/15

Thus, MS4Al2 is a cell membrane-located differentiation antigen fornormal colon epithelia, which is also expressed in colorectal tumors andmetastases.

Example 5 Identification of BRCO1 as a New Tumor Antigen

BRCO1 and its translation product have not been described previously.The datamining method of the invention produced the EST (expressedsequence tag) AI668620. RT-PCR studies using specific primers (sense:CTTGCTCTGAGTCATCAGATG, (SEQ ID NO:46) antisense: CACAGAATATGAGCCATACAG)(SEQ ID NO:47) were carried for expression analysis. According to theinvention, specific expression was found in testicular tissue andadditionally in normal mammary gland (Table 5). In all other tissues,this antigen is transcriptionally repressed. It is likewise detected inmammary gland tumors (20 out of 20). BRCO1 is distinctly overexpressedin breast tumors in comparison with expression in normal mammary glandtissue (FIG. 10). Utilizing EST contigs (the following ESTs wereincorporated: AW137203, BF327792, BF327797, BE069044, BF330665), morethan 1500 bp of this transcript were cloned according to the inventionby electronic full-length cloning (SEQ ID NO:39). The sequence maps tochromosome 10p11-12. In the same region, in immediate proximity, thegene for a mammary differentiation antigen, NY-BR-1, has been describedpreviously (NM 052997; Jager, D. et al., Cancer Res. 61(5):2055-61,2001).

TABLE 5 Expression of BRCO1 in normal tissues and breast tumors Ileum −Colon − Liver − Lung − Lymph nodes − Stomach − Spleen − Adrenal gland −Kidney − Esophagus − Ovary − Rectum − Testis + Thymus − Skin − Mamma +Pancreas − PBMC − PBMC act. − Prostate − Thyroid − Tube − Uterus −Cerebrum − Cerebellum − Mammary carcinomas ++ (20/20)

Matched pair (mammary carcinoma and adjacent normal tissue) studiesrevealed BRCO1 overexpression in 70% of the mammary carcinomas incomparison with the normal tissue.

Thus, BRCO1 is a new differentiation antigen for normal mammary glandepithelia, which is overexpressed in breast tumors.

Example 6 Identification of TPX1 as a New Tumor Antigen

The sequence of TPX1 (Acc. No. NM_003296; SEQ ID NO: 40) and of itstranslation product (SEQ ID NO:41, are known. The antigen has beendescribed previously only as being testis-specific, that is as anelement of the outer fibers and of the acrosome of sperms. Previously,an involvement as adhesion molecule in the attachment of sperms toSertoli cells has been attributed to said antigen (O'Bryan, M. K. etal., Mol. Reprod. Dev. 58(1):116-25, 2001; Maeda, T, et al., Dev. GrowthDiffer. 41(6):715-22, 1999). The invention reveals, for the first time,aberrant expression of TPX1 in solid tumors (Table 6). Owing to themarked amino acid homology between TPX1 and the neutrophile-specificmatrix glycoprotein SGP 28 (Kieldsen et al., FEBS Lett 380:246-259,1996), TPX1-specific protein sequences comprising the peptide SREVTTNAQR(SEQ ID NO:84) are suitable according to the invention for preparingdiagnostic and therapeutic molecules.

TABLE 6 Expression of TPX1 in tumors Tested in Tissue total Positive %Melanoma 16 1 6 Mammary carcinomas 20 3 15 Colorectal tumors 20 0 0Prostate carcinomas 8 3 37 Bronchial carcinomas 17 2 11 Kidney cellcarcinomas 7 1 14 Ovarian carcinomas 7 1 14 Thyroid carcinomas 4 0 0Cervical carcinomas 6 1 16 Melanoma cell lines 8 2 25 Bronchialcarcinoma cell lines 6 1 16

Example 7 Identification of BRCO2 as a New Tumor Genetic Product

BROC2 and its translation product have not been described previously.The method of the invention produced the ESTs (expressed sequence tag)BE069341, BF330573 and AA601511. RT-PCR studies using specific primers(sense: AGACATGGCTCAGATGTGCAG, (SEQ ID NO:66) antisense:GGAAATTAGCAAGGCTCTCGC) (SEQ ID NO:67) were carried out for expressionanalysis. According to the invention, specific expression was found intesticular tissue and additionally in normal mammary gland (Table 7). Inall other tissues, this genetic product is transcriptionally repressed.It is likewise detected in mammary gland tumors. Utilizing EST contigs(the following ESTs were incorporated: BF330573, AL044891 and AA601511),1300 bp of this transcript were cloned according to the invention byelectronic full-length cloning (SEQ ID 62), The sequence maps tochromosome 10p11-12. In the same region, in immediate proximity, thegene for a mammary differentiation genetic product, NY-BR-1, has beendescribed previously (NM_052997; Jager, D. et al., Cancer Res.61(5):2055-61, 2001), and here the BRCO1 described above under Example 6is located. Further genetic analyses revealed according to the inventionthat the sequence listed under SEQ ID NO:62 represents the 3′untranslated region of the NY-BR-1 gene, which has not been describedpreviously.

TABLE 7 Expression of BRCO2 in normal tissues and breast tumors TissueExpression Testis + Mamma + Skin − Liver − Prostate − Thymus − Brain −Lung − Lymph nodes − Spleen − Adrenal gland − Ovary − Leukocytes − Colon− Esophagus − Uterus − Skeleton muscle − Epididymis − Bladder − Kidney −Mammary carcinoma +

BRCO2 is a new differentiation genetic product for normal mammary glandepithelia, which is also expressed in breast tumors.

Example 8 Identification of PCSC as a New Tumor Genetic Product

PCSC (SEQ ID NO:63) and its translation product have not been describedpreviously. The datamining method of the invention produced the EST(expressed sequence tag) BF064073. RT-PCR studies using specific primers(sense: TCAGGTATTCCCTGCTCTTAC, (SEQ ID NO:68) antisense:TGGGCAATTCTCTCAGGCTTG) (SEQ ID NO:69) were carried out for expressionanalysis. According to the invention, specific expression was found innormal colon, and additionally in colon carcinomas (Table 5). In allother tissues, this genetic product is transcriptionally repressed. PCSCcodes for two putative ORFs (SEQ ID 64 and SEQ ID 65) Sequence analysisof SEQ ID 64 revealed a structural homology to CXC cytokines. Inaddition, 4 alternative PCSC cDNA fragments were cloned (SEQ IDNO:85-88). In each case, according to the invention, each cDNA contains3 putative ORFs which code for the polypeptides depicted in SEQ IDNO:89-100.

TABLE 8 Expression of PCSC in normal tissues and colorectal carcinomasIleum + Colon + Liver − Lung − Lymph nodes − Stomach − Spleen − Adrenalgland − Kidney − Esophagus − Ovary − Rectum + Testis − Thymus − Skin −Mamma − Pancreas − PBMC − PBMC act. − Prostate − Thyroid − Tube − Uterus− Cerebrum − Cerebellum − Colorectal tumors 19/20 Colorectal tumors15/15 metastases

Thus, PCSC is a differentiation antigen for normal colon epithelia whichis also expressed in colorectal tumors and in all colon metastasesstudied. PCSC expression detected in all colorectal metastases accordingto the invention renders this tumor antigen a very interesting targetfor prophylaxis and treatment of metastasizing colon tumors.

Example 9 Identification of SGY-1 as a New Tumor Antigen

The sequences of the SGY-1 transcript (SEQ ID NO:70) and of itstranslation product (SEQ ID NO:71) have been published in GenBank underaccession number AF177398 (Krupnik et al., Gene 238, 301-313, 1999).Soggy-1 has previously been described as a member of the Dickkopfprotein family which act as inhibitors and antagonists of the Wnt familyof proteins. The Wnt proteins in turn have important functions inembryonic development. Based on the sequence of SGY-1 (SEQ ID NO:70),PCR primers (5′-CTCCTATCCATGATGCTGACG-3′ (SEQ ID NO:72) and5′-CCTGAGGATGTACAGTAAGTG-3′) (SEQ ID NO:73) were generated according tothe invention and used for RT-PCR analyses (95° 15 min; 94° 1 min; 63° 1min; 72° 1 min; 35 cycles) in a number of human tissues. Expression innormal tissues was shown to be limited to testis. As described for theother eCT, SGY-1 was shown according to the invention to be ectopicallyactivated in a number of tumor tissues; cf. Table 9.

TABLE 9 Expression of SGY-1 in tumors Tested Tissue in total Positive %Melanoma 16 4 25 Mammary carcinomas 20 4 20 Colorectal tumors 20 0 0Prostate carcinomas 8 1 13 Bronchial carcinomas 32 3 18 Kidney cellcarcinomas 7 0 0 Ovarian carcinomas 7 4 57 Thyroid carcinomas 4 0 0Cervical carcinomas 6 2 33 Melanoma cell lines 8 2 25 Bronchialcarcinoma cell lines 6 2 33 Mammalian carcinoma cell lines

Example 10 Identification of MORC as a New Tumor Antigen

The sequences of the MORC transcript (SEQ ID NO:74) and of itstranslation product (SEQ ID NO:75) have been published in GenBank underthe accession number XM_037008 (Inoue et al., Hum Mol Genet. July:8(7):1201-7, 1999).

MORC has originally been described as being involved in spermatogenesis.Mutation of this protein in the mouse system results in underdevelopmentof the gonads.

Based on the sequence of MORC (SEQ ID NO:74), PCR primers(5′-CTGAGTATCAGCTACCATCAG-3′ (SEQ ID NO:76) and5′-TCTGTAGTCCTTCACATATCG-3′) (SEQ ID NO:77) were generated according tothe invention and used for RT-PCR analyses (95° 15 min; 94° 1 min; 63° 1min; 72° 1 min; 35 cycles) in a number of human tissues. Expression innormal tissues was shown to be limited to testis. As described for theother eCT, MORC was shown according to the invention to be ectopicallyactivated in a number of tumor tissues cf. Table 10.

TABLE 10 Expression of MORC in tumors Tested in Tissue total Positive %Melanoma 16 3 18 Mammary carcinomas 20 0 0 Colorectal tumors 20 0 0Prostate carcinomas 8 0 0 Bronchial carcinomas 17 3 18 Kidney cellcarcinomas 7 0 0 Ovarian carcinomas 7 1 14 Thyroid carcinomas 4 0 0Cervical carcinomas 6 0 0 Melanoma cell lines 8 1 12 Bronchial carcinomacell lines 6 1 17

The invention claimed is:
 1. A method of diagnosing cancer, which methodcomprises detection of a nucleic acid which codes for a tumor-associatedantigen in a biological sample isolated from a patient, with saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 19-21, 54 and 56 wherein the detectioncomprises (i) contacting the nucleic acids isolated from the biologicalsample with a primer set comprising SEQ ID NO: 27 and SEQ ID NO: 28under conditions which amplifies the nucleic acids; wherein theconditions are suitable for RT-PCR comprising incubating the primer setand the nucleic acids at about 95° C. for about 15 minutes, incubatingthe primer set and the nucleic acids at about 94° C. for about 1 minute,hybridizing the primer set to the nucleic acids at about 63° C. forabout 1 minute, and amplifying the nucleic acids at about 72° C. forabout 1 minute, and repeating for about 35 cycles; and (ii) detectingthe amplified nucleic acid; wherein an increased or abnormal amount ofthe nucleic acid in the biological sample as compared to a normalbiological sample without cancer indicates the patient has cancer; andwherein the cancer is selected from the group consisting of seminomas,melanoma, teratomas, gliomas, colorectal cancer, breast cancer, prostatecancer, cancer of the uterus, ovarian cancer, lung cancer, kidneycancer, thyroid cancer, cervical cancer, and carcinomas.
 2. The methodof claim 1, wherein the tumor-associated antigen comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 22-24, 58 and60.
 3. A method for determining regression, course, or onset of cancer,which method comprises monitoring a sample from a patient who has canceror is suspected of falling ill with cancer, with respect to the amountof nucleic acid which codes for the tumor-associated antigen, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 19-21, 54 and 56 wherein the monitoring isdetermined in a first sample at a first point in time and in a secondsample at a second point in time wherein the monitoring comprises (i)contacting the nucleic acid isolated from the sample with a primer setcomprising SEQ ID NO: 27 and SEQ ID NO: 28 under conditions whichamplifies the nucleic acids; wherein the conditions are suitable forRT-PCR comprising incubating the primer set and the nucleic acids atabout 95° C. for about 15 minutes, incubating the primer set and thenucleic acids at about 94° C. for about 1 minute, hybridizing the primerset to the nucleic acids at about 63° C. for about 1 minute, andamplifying the nucleic acids at about 72° C. for about 1 minute, andrepeating for about 35 cycles; and (ii) detecting the amplified nucleicacid; wherein an increased or abnormal amount of the nucleic acid in thefirst sample as compared to the second sample indicates the regression,course, or onset of cancer; and wherein the cancer is selected from thegroup consisting of seminomas, melanoma, teratomas, gliomas, colorectalcancer, breast cancer, prostate cancer, cancer of the uterus, ovariancancer, lung cancer, kidney cancer, thyroid cancer, cervical cancer, andcarcinomas.
 4. The method of claim 3, in which the tumor-associatedantigen comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22-24, 58 and
 60. 5. A method of diagnosingcancer, which method comprises detection of a nucleic acid which codesfor a tumor-associated antigen in a biological sample isolated from apatient, with said tumor-associated antigen having a sequence encoded bya nucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 19-21, 54 and 56; wherein the detectioncomprises (i) contacting the nucleic acid isolated from the biologicalsample with a probe prepared by amplification with a primer setcomprising SEQ ID NO: 27 and SEQ ID NO: 28 under conditions that allowthe probe to hybridize to the nucleic acid; (ii) detecting a complexformed between the probe and the nucleic acid from the sample; whereinan increased or abnormal amount of the complex comprising the nucleicacid in the biological sample as compared to a normal biological sampleindicates the patient has cancer; and wherein the cancer is selectedfrom the group consisting of seminomas, melanoma, teratomas, gliomas,colorectal cancer, breast cancer, prostate cancer, cancer of the uterus,ovarian cancer, lung cancer, kidney cancer, thyroid cancer, cervicalcancer, and carcinomas.
 6. The method of claim 5, in which thetumor-associated antigen comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22-24, 58 and
 60. 7. A method fordetermining regression, course, or onset of cancer, which methodcomprises monitoring a sample from a patient who has cancer or issuspected of falling ill with cancer, with respect to the amount ofnucleic acid which codes for the tumor-associated antigen, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 19-21, 54 and 56; wherein the monitoring isdetermined in a first sample at a first point in time and in a secondsample at a second point in time; wherein the monitoring comprises (i)contacting the nucleic acid isolated from the sample with a probeprepared by amplification with a primer set comprising SEQ ID NO: 27 andSEQ ID NO: 28 under conditions that allow the probe to hybridize to thenucleic acid; (ii) detecting a complex formed between the probe and thenucleic acid from the sample; wherein an increased or abnormal amount ofthe complex comprising the nucleic acid in the first sample as comparedto the second sample indicates the regression, course, or onset ofcancer; and wherein the cancer is selected from the group consisting ofseminomas, melanoma, teratomas, gliomas, colorectal cancer, breastcancer, prostate cancer, cancer of the uterus, ovarian cancer, lungcancer, kidney cancer, thyroid cancer, cervical cancer, and carcinomas.8. The method of claim 7, in which the tumor-associated antigencomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 22-24, 58 and 60.